Fig. 1

Residual testing of heregulin in the manufacture of hSCs for transplantation. The diagram outlines the workflow used to test heregulin activity directly using aliquots of culture medium (panel 1). In these assays, mitogen- and serum-starved rat SCs (panel 2) were used for their capacity to effectively bind and transduce signals from heregulin-activated ErbB2/3 (inset in panel 2). Detection was carried out using antibodies against the total and phosphorylated forms of ErbB3, ERK and Akt followed by incubation with HRP-conjugated secondary antibodies. Immunoreactive bands were visualized by capturing the product from an ECL reaction on light-sensitive film. Quantification of the Western blot data was done by densitometric analysis using ImageJ. The total operating time from onset (plating of the rat SCs) to completion (data analysis) was 4–5 days on average. An example of P-ERK and T-ERK detection and quantification from a typical gel is shown in panel 3. The ERK signal is revealed as a doublet consisting of closely related isoforms ERK1 and ERK2 (panel 3)